RT-qPCR-based tests for SARS-CoV-2 detection in pooled saliva samples for massive population screening to monitor epidemics

Abstract

Swab, RT-qPCR tests remain the gold standard of diagnostics of SARS-CoV-2 infections. These tests are costly and have limited throughput. We developed a 3-gene, seminested RT-qPCR test with SYBR green-based detection designed to be oversensitive rather than overspecific for high-throughput diagnostics of populations. This two-tier approach depends on decentralized self-collection of saliva samples, pooling, 1st-tier testing with highly sensitive screening test and subsequent 2nd-tier testing of individual samples from positive pools with the IVD test. The screening test was able to detect five copies of the viral genome in 10 µl of isolated RNA with 50% probability and 18.8 copies with 95% probability and reached Ct values that were highly linearly RNA concentration-dependent. In the side-by-side comparison, the screening test attained slightly better results than the commercially available IVD-certified RT-qPCR diagnostic test DiaPlexQ (100% specificity and 89.8% sensitivity vs. 100% and 73.5%, respectively). Testing of 1475 individual clinical samples pooled in 374 pools of four revealed 0.8% false positive pools and no false negative pools. In weekly prophylactic testing of 113 people within 6 months, a two-tier testing approach enabled the detection of 18 infected individuals, including several asymptomatic individuals, with substantially lower cost than individual RT-PCR testing.

Pooled RNA-extraction-free testing of saliva for the detection of SARS-CoV-2

“…However, despite the resource-saving benefits 14 , 15 , 18 , 22 , clinical laboratories have been hesitant to implement pooled sample testing 23 due to: (1) stringent workflows which do not fit within existing laboratory operations, (2) a lack of clear guidance on how to implement such methods and (3) the perception that clinical sensitivity of the assay will be lost with pooling. The methods we propose in the current study are straightforward extensions of a simple SARS-CoV-2 testing method and can be easily conducted manually, without requiring additional investment.…”

“…Large pooled testing programs have successfully demonstrated the efficacy of pooled saliva testing for helping to keep schools safely open 15 , 29 , with pooled samples having a similar sensitivity to the molecular testing of individual samples, in terms of both qualitative and quantitative (comparable Ct values between pooled and individual samples) measures. It is important to note however, that during times of high virus prevalence, when a greater number of tests are expected to return a positive result, pooled testing strategies must be frequently reviewed and when necessary, updated to minimize the number of reflex tests required which can negate the resource-savings benefits of pooled testing 13 , 19 , 22 , 27 .…”

Development and Optimization of a Multiplex Real-Time RT-PCR to Detect SARS-CoV-2 in Human Samples

“…This highlights the need to implement rapid and specific techniques to efficiently detect SARS-CoV-2. Molecular tests such as reverse transcription-polymerase chain reaction (RT-PCR) and its quantitative variant RT-qPCR are excellent diagnostic options, given their ability to detect target nucleic acids with high sensitivity [ 2 ] ( Figure S1 ).…”

A one-step low-cost molecular test for SARS-CoV-2 detection suitable for community testing using minimally processed saliva

“…One advantage of these tests, as opposed to RT-PCR with fluorogenic probes, is that they allow for assessment of the amplification specificity by simply examining the amplicon melting curves at the end of the PCR [ 28 ]. While most SYBR Green-based RT-PCR assays have been established for naso or oropharyngeal specimens [ 16 , 18–27 ], a handful of studies showed promising evidence for using saliva samples in these tests [ 17 , 29–31 ]. However, all the protocols described thus far require prior viral RNA isolation, which increases the costs and time needed for testing.…”